Dynamic organelle membrane contacts as key regulators of virus replication

动态细胞器膜接触作为病毒复制的关键调节因子

• 批准号: 10011555
• 负责人: Katelyn Camille Cook
• 金额: $ 4.55万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011555
• 关键词: Apoptosis; Biochemical; Biogenesis; Biological; Biological Assay; Biological Models; Biological Process; Biology; Biotin; Cells; Cellular biology; Cholesterol; Complex; Confocal Microscopy; Cytomegalovirus; Cytomegalovirus Infections; Endoplasmic Reticulum; Endosomes; Eukaryotic Cell; Future; Gene Expression; Genetic Transcription; Growth; Herpes Simplex Infections; Herpesviridae; Herpesvirus 1; Human; Image; Infection; Influenza A virus; Knowledge; Label; Ligase; Link; Lipids; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Metabolism; Methods; Microscopy; Mitochondria; Molecular Virology; Organelles; Orthomyxoviridae; Phenotype; Pilot Projects; Plasmalogens; Play; Process; Production; Proteins; Proteomics; Regulation; Role; Series; Site; Small Interfering RNA; Specificity; Stimulus; Stress; Structure; Subcellular Spaces; Testing; Time; Up-Regulation; Vesicle; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Work; base; cholesterol control; endosome membrane; human pathogen; immunoregulation; knock-down; live cell imaging; live cell microscopy; obligate intracellular parasite; particle; peroxisome; peroxisome membrane; stem; trafficking; virology

PROJECT ABSTRACT Cellular organelles form dynamic intracellular networks by engaging in direct membrane contact. Membrane contact sites (MCSs), which link organelle membranes via protein interactions, are fundamental to the regulation of organelle structure, composition, and dynamics. As these features dictate organelle function, MCSs also control diverse biological processes like, metabolism, trafficking, organelle biogenesis, and apoptosis. However, organelle contacts remain largely unexplored during mammalian virus infection despite the knowledge that organelles play critical, and often conserved, roles in a broad range of virus infections. Moreover, many virus- induced changes to organelles, such as vesicular remodeling, mitochondrial fragmentation, and immune modulation, are directly related to MCS-controlled functions. I hypothesize that membrane contact sites are key regulatory hubs of virus replication and actively modulated during infection. I propose an integrative live-cell imaging and quantitative proteomics approach to examine MCS dynamics across subcellular space and infection time. This workflow includes targeted proteomics to determine temporal MCS protein abundances, confocal microscopy to examine organelle contact phenotypes, and molecular virology-based functional assays that delve into the mechanisms underlying the roles of membrane contact sites in virus replication. I have already established an experimental method to track alterations in MCS protein abundances with high sensitivity and precision during the progression of a viral infection. I further applied this method in infections with several human viruses, including the herpesviruses cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1), and the orthomyxovirus Influenza A. Indeed, I discovered that these viruses change the composition of MCSs across infection time, with temporal and organelle specificity related to each unique virus replication strategy. Among these viruses, the wide-spread human pathogen HCMV caused the most striking changes, triggering a nearly global upregulation and rewiring of MCSs. Given the known role of the endoplasmic reticulum (ER) as a master regulator of cellular organelles, ER-mediated contacts are poised to facilitate key steps in the virus replication cycle, and my preliminary functional analyses support this. In this study, I will investigate the function and regulation of two critical ER contacts in HCMV infection. First, I will establish the role of ER-peroxisome contacts in modulating peroxisome plasticity for virus assembly. I hypothesize that ER-peroxisome contact is increased during infection and modified by virus-host protein interactions to enhance lipid synthesis for formation of the viral envelope. Second, I will investigate ER-endosome contacts as regulators of the vesicular rearrangements required for virion assembly and egress, which I predict occurs in an endosome type-specific manner to control cholesterol distribution and endosome trafficking directionality. As MCSs are fundamental to organelle biology, this work will help expand the understanding of the mechanisms at the core of human virus replication.

项目摘要 细胞器通过直接膜接触形成动态的细胞内网络。膜 接触位点（MCS）通过蛋白质相互作用连接细胞器膜，是调节的基础 细胞器的结构、组成和动力学。由于这些特征决定了细胞器的功能， 控制不同的生物过程，如代谢、运输、细胞器生物发生和凋亡。然而，在这方面， 在哺乳动物病毒感染期间，细胞器接触仍然在很大程度上未被探索， 细胞器在广泛的病毒感染中起着关键的、通常是保守的作用。此外，许多病毒- 诱导细胞器的变化，如囊泡重塑，线粒体断裂，免疫 调制直接与MCS控制功能相关。我假设膜接触部位 病毒复制的关键调控中心，并在感染期间积极调节。我建议一个综合的 活细胞成像和定量蛋白质组学方法来检查跨亚细胞空间的MCS动态， 感染时间该工作流程包括靶向蛋白质组学以确定时间MCS蛋白质丰度， 共聚焦显微镜检查细胞器接触表型，以及基于分子病毒学的功能测定 深入研究了病毒复制中膜接触位点的作用机制。我已经 建立了高灵敏度跟踪MCS蛋白丰度变化的实验方法， 在病毒感染过程中的精确度。我进一步将这种方法应用于几种人类感染， 病毒，包括疱疹病毒巨细胞病毒（HCMV）和单纯疱疹病毒1（HSV-1），以及 甲型流感病毒事实上，我发现这些病毒改变了MCSs的组成， 感染时间，与时间和细胞器特异性相关的每一个独特的病毒复制策略。之间 在这些病毒中，广泛传播的人类病原体HCMV引起了最引人注目的变化，引发了近 MCSs的整体上调和重新布线。鉴于已知内质网（ER）作为主细胞的作用， 作为细胞器的调节器，ER介导的接触有助于病毒复制的关键步骤 循环，我的初步功能分析支持这一点。在这项研究中，我将研究功能和 调节HCMV感染中两个关键的ER接触。首先，我将建立ER-过氧化物酶体接触的作用， 在调节过氧化物酶体可塑性的病毒装配。我假设ER-过氧化物酶体接触增加 在感染过程中，通过病毒-宿主蛋白质相互作用进行修饰，以增强脂质合成， 病毒包膜其次，我将研究ER-内体接触作为囊泡重排的调节剂， 这是病毒体组装和出口所必需的，我预测这是以一种内体类型特异性的方式发生的， 胆固醇分布和内体运输方向性。由于MCS是细胞器生物学的基础， 这项工作将有助于扩大对人类病毒复制核心机制的理解。